| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Reproductive Endocrinology Center, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, California 94143-0556
Address all correspondence and requests for reprints to: Robert B. Jaffe, M.D., Reproductive Endocrinology Center, Department of Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco, San Francisco, California 94143-0556. E-mail: robert_jaffe{at}quickmail.ucsf.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To date, no studies of phytoestrogen actions on adrenal function have been reported. However, we and others previously found that steroid production by human fetal adrenal cortical cells is modulated by estrogen (20, 21, 22). In cultured human fetal adrenal cortical cells, 17ß-estradiol at relatively high concentrations (1–10 µmol/L), augmented corticotropin (ACTH)-stimulated androgen [mainly dehydroepiandrosterone sulfate (DHEA-S)] production, whereas glucocorticoid (cortisol) synthesis was significantly inhibited (22). Although these observations showed that in vitro, adrenal cortical cell function can be modulated by estrogen, their physiological significance is unclear, as endogenous estrogen concentrations do not normally reach micromole per L levels in nonpregnant adults. However, it is possible that exogenous estrogen-related compounds, such as dietary phytoestrogens, could reach circulating levels high enough to exert estrogenic actions on the adrenal cortex. This is particularly the case in infants and adults who consume large amounts of soy-derived foods. For example, circulating concentrations of total phytoestrogens as a result of the typical Japanese diet are in the 1–5 µmol/L range (23, 24), and in infants fed exclusively soy-based formula, plasma genistein and daidzein concentrations are approximately 4 µmol/L, 10,000–20,000 times higher than plasma 17ß-estradiol concentrations (25). More importantly, on a per weight basis, levels of genistein and daidzein in those infants were an order of magnitude higher than typical plasma concentrations in adults consuming soy foods, suggesting that young children may be particularly susceptible to phytoestrogenic actions. Thus, we hypothesized that the human adrenal cortex is susceptible to functional modulation by dietary phytoestrogens. Therefore, we examined the effects of two phytoestrogens, genistein and daidzein, on basal and ACTH-stimulated steroid production by human fetal and postnatal adrenocortical cell cultures and in the human adult adrenal cortical cell line, H295R.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human fetal adrenal glands were obtained from second trimester
fetuses (16–23 weeks of gestation as assessed by foot length) after
elective therapeutic termination of pregnancy by dilatation and
evacuation. A pair of human postnatal (
1.5 yr of age) adrenals were
obtained from an organ donor through the kidney transplant service at
the University of California-San Francisco (UCSF). The study protocol
was approved by the committee on human research, UCSF. The H295R cell
line was provided by Dr. W. Rainey, University of Texas Southwestern
Medical Center (Dallas, TX). Genistein and daidzein were obtained from
Calbiochem (San Diego, CA). Human ACTH [ACTH-(1–24)]
was obtained from Organon (West Orange, NJ). Progesterone,
17
-hydroxyprogesterone, 11-deoxycortisol, 17ß-estradiol, and cAMP
were obtained from Sigma Chemical Co. (St. Louis, MO). The
full-length complementary DNAs (cDNAs) encoding human cytochrome P450
cholesterol side-chain cleavage (P450scc), P450 17-hydroxylase/17,20
lyase (P450c17), and P450 21-hydroxylase (P450c21) were provided by Dr.
W. L. Miller, UCSF (26, 27, 28); human type II 3ß-hydroxysteroid
dehydrogenase/isomerase (3ßHSD) was provided Dr. F. Labrie,
University of Laval (Quebec, Canada) (29); human P450 11ß-hydroxylase
(P450c11) was provided by Dr. P. White, University of Texas
Southwestern Medical School (30), and human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was obtained from American Type Culture Collection (Manassas, VA).
Cell cultures
Primary cultures of human fetal and postnatal adrenocortical cells were prepared as previously described (31). Briefly, the adrenal capsule was carefully peeled away from the gland, and the remaining cortical cells were dispersed by enzymatic digestion and plated onto 6-cm diameter plastic culture dishes (Falcon Plastics, Los Angeles, CA) at a density of 5 x 105 cells/dish or onto 48-well culture dishes (Nunc, Naperville, IL) at a density of 1 x 105 cells/well. In each case, the culture medium was DMEM H-16/Ham’s F-12 (1:1) medium containing nonessential amino acids and supplemented with 10% FCS, 2 mmol/L glutamine, and 50 µg/mL gentamicin (Cell Culture Facility, UCSF). After 48 h, test substances were added. H295R cells were plated onto 48-well dishes in DMEM H-16/Ham’s F-12 (1:1) medium supplemented with 2 mmol/L glutamine, 50 µg/mL gentamicin, 2% FCS, and 1% ITS Plus (insulin, transferrin, and selenium, each 6.25 µg/mL; Collaborative Research, Bedford, MA) and grown to confluence. All plates were incubated in a humidified environment at 37 C in 95% air-5% CO2.
Ribonucleic acid (RNA) analysis
Northern blot analyses were used to assay the abundance of specific messenger RNA (mRNA) transcripts. Total RNA was extracted and purified from cultured cells using the method of Chomczynski and Sacchi (32). Total RNA (5–10 µg) was denatured in formaldehyde, subjected to electrophoresis through a 1.2% agarose gel, and transferred to nitrocellulose membranes (Nytran, Schleicher & Schuell, Inc., Keene, NH) to which it was cross-linked by exposure to UV light (Stratalinker, Stratagene, La Jolla, CA). [32P]deoxy-CTP-labeled cDNA probes (1–2 x 109 dpm/µg) were synthesized by random primer extension of denatured full-length cDNAs. Prehybridization was performed in hybridization buffer (Quickhyb buffer, Stratagene) at 68 C for 15 min. Denatured radiolabeled probe was then added to the membranes and incubated at 68 C for 1 h. To remove nonspecifically bound probe, membranes were washed in 2 x SSC (1 x SSC is 0.15 mol/L NaCl and 0.015 mol/L sodium citrate)-0.1% SDS at room temperature for 15 min and then in 0.1 x SSC-0.1% SDS at 60 C for 30 min. Membranes were then subjected to autoradiography at -70 C. The relative abundance of mRNA transcripts was estimated by computer-assisted densitometry (BioImage, Ann Arbor, MI). All data were normalized to the abundance of transcripts encoding GAPDH, which was constitutively expressed. Probes were removed by washing the membranes in distilled water at 100 C. Complete removal of probe was confirmed by autoradiography before reprobing.
RIAs
Cortisol, DHEA, and DHEA-S were measured in conditioned medium using specific RIAs. Unconjugated cortisol was assayed using a kit purchased from Diagnostic Products (Los Angeles, CA). DHEA and DHEA-S were assayed using specific kits obtained from ICN Biomedicals, Inc. (Costa Mesa, CA), with charcoal separation of free from bound steroid. All assays were validated for use on conditioned medium from fetal adrenal cortical cell cultures, and each had an inter- and intraassay coefficient of variation of less than 10%.
Statistical analyses
All steroid data were normalized for cell number [measured using a particle counter (Coulter Electronics, Hialeah, FL)] and time of exposure to ACTH and are presented as the mean ± SEM. All experiments were performed in triplicate and repeated at least three times. Statistical analyses were conducted by ANOVA followed by post-hoc tests (Dunnett’s, Student-Newman-Keuls, or Bonferroni), and differences were considered statistically significant when P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In all cell types studied, genistein and daidzein decreased
agonist (ACTH or cAMP)-stimulated cortisol production in a
dose-dependent fashion with an ED50 of 1–4 µmol/L (Fig. 1
, A and B, upper panels). At
the maximum phytoestrogen dose (40 µmol/L), cortisol production was
decreased to levels approaching the lower limit of assay detection in
all cell types. In postnatal and H295R cells, genistein and daidzein
also decreased basal cortisol production in a similar dose-dependent
fashion. In fetal adrenal cortical cells, basal cortisol production
was close to the lower limit of assay detection; therefore, any further
decrease induced by phytoestrogens was not apparent.
|
Steroidogenic enzyme expression
To examine the mechanism by which phytoestrogens modulate steroid
production by adrenal cortical cells, we determined whether genistein
and daidzein alter the expression of the steroid-metabolizing enzymes
involved in DHEA and cortisol synthesis by human fetal
adrenal cortical cells. The synthesis of cortisol from pregnenolone
(Fig. 2A) is performed sequentially by
four enzymes: 3ßHSD, P450c17, P450c21, and P450c11, whereas
DHEA production requires only P450scc and P450c17. The
effects of 24-h exposure of human fetal adrenal cortical cells to
genistein and daidzein (both 40 µmol/L) in the presence and absence
of ACTH on the expression of genes encoding these enzymes were examined
by Northern blot analysis. Relative to the abundance of mRNA encoding
the constitutively expressed GAPDH, genistein and daidzein did not
alter the basal or ACTH-stimulated abundance of mRNAs encoding P450scc,
3ßHSD, P450c17, P450c21, or P450c11. In the representative experiment
shown in Fig. 2B, genistein and daidzein appeared to decrease 3ßHSD
mRNA; however, this effect was not reproducible (n = 3).
|
We next examined whether the phytoestrogens modulate steroidogenic
enzyme activity. To that end, fetal adrenal cortical cells were exposed
for 24 h to genistein or daidzein with and without ACTH and in the
presence of specific steroid precursors of cortisol synthesis. The
rationale underlying these experiments was that by providing different
substrates we would bypass, and therefore identify, any step in the
pathway (Fig. 2A
) toward cortisol synthesis affected by the
phytoestrogens. Both genistein and daidzein inhibited ACTH-stimulated
cortisol production when progesterone (Fig. 3B) and 17-hydroxyprogesterone (Fig. 3C) were added to the medium. However, when 11-deoxycortisol was added
to the cultures, neither genistein nor daidzein inhibited cortisol
production (Fig. 3D). Similar results were obtained using H295R cells.
Taken together, these data suggest that phytoestrogens suppress
cortisol production by inhibiting P450c21.
|
Genistein and daidzein (each at 40 µmol/L) were not toxic to human adrenal cortical cells, as assessed by cell number, and their effects were transient; exposure to genistein or daidzein for 24 h had no effect on the subsequent response to ACTH (data not shown). Thus, at the maximum concentrations studied, the phytoestrogens did not permanently alter the cell’s steroidogenic phenotype. As genistein inhibits protein tyrosine kinase activity (33), we determined whether its effects were due to inhibition of tyrosine phosphorylation. By Western blot analysis, we found that genistein did not alter the spectrum of peptides containing phosphotyrosine residues in human fetal adrenal cortical cells (data not shown). That the actions of daidzein, which does not alter tyrosine kinase activity, were identical to those of genistein further indicates that the actions of genistein were not mediated by inhibition of tyrosine kinase activity.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our data indicate that genistein and daidzein decreased cortisol
synthesis by suppressing P450c21 enzymatic activity; expression of this
gene was not affected. Although 3ßHSD activity was not assessed in
the present studies, it is likely, based on previous findings, that
genistein and daidzein inhibited cortisol synthesis by decreasing the
activity of 3ßHSD. Studies in human adult adrenal microsomes (34) and
in the H295R cell line (35) have demonstrated that estrogens inhibit
cortisol synthesis by specifically inhibiting the activity of the
3ßHSD enzyme. The effects of phytoestrogens on H295R cells reported
here were essentially identical to the effects of estradiol on H295R
cells reported by Gell et al. (35). Interestingly, Gell
et al. (35) also showed that the actions of estradiol on
steroid production by H295R cells were not inhibited by the estrogen
antagonist, ICI 182,780, suggesting that the estrogen effects were not
mediated through estrogen receptors. Although Kuiper et al.
(2) showed that phytoestrogens, including genistein and daidzein,
compete with estradiol for binding to estrogen receptors (both the -
and ß-subtypes) and regulate estrogen-responsive gene expression,
these processes do not appear to be involved in the
estrogen/phytoestrogen modulation of adrenal cortical cell
steroidogenesis. We previously showed that estradiol inhibits cortisol
synthesis by human fetal adrenal cortical cells without influencing the
expression of P450scc, P450c17, and 3ßHSD (22). Similarly, in the
present study, phytoestrogens altered steroid production without
affecting the expression of steroidogenic enzymes. Taken together,
these findings support the concept that estrogen and phytoestrogens
modulate adrenal steroidogenic activity via nongenomic mechanisms, most
likely by direct modulation of steroidogenic enzyme activity.
As well as decreasing cortisol synthesis, genistein and daidzein modulated androgen production by increasing DHEA and DHEA-S synthesis. However, effects on androgen synthesis occurred only in postnatal adrenal cortical cells and in the H295 cell line; in fetal adrenal cortical cells, basal and ACTH-stimulated DHEA-S production were not altered by exposure to phytoestrogens. The mechanism by which the phytoestrogens increased androgen production is not clear. As with the decrease in cortisol production, it is possible that genistein and daidzein increased DHEA and DHEA-S production by directly modulating steroidogenic enzyme activity, i.e. by augmenting the activity of either P450scc or P450c17. However, the abundance of mRNAs encoding P450scc and P450c17 was not affected by genistein or daidzein. Furthermore, genistein and daidzein did not affect basal or ACTH-stimulated production of DHEA-S in fetal adrenal cortical cells, suggesting that the activities of these enzymes were not affected. It is more likely that androgen production was increased as a result of decreased cortisol synthesis, i.e. the block in the cortisol biosynthetic pathway caused an increase in the pool of precursors available for DHEA synthesis. Interestingly, basal DHEA and DHEA-S production was increased only in cells in which basal cortisol production was high. In postnatal adrenal cortical cells, genistein increased DHEA and DHEA-S production in a dose-responsive fashion, which mirrored the decrease in cortisol, suggesting that the two events were related.
The possible consequences of altered adrenal function by dietary phytoestrogens are intriguing. Suppression of cortisol production is not likely to have a major impact, as the hypothalamic-pituitary-adrenal axis is exquisitely sensitive to cortisol negative feedback and therefore would be expected to accommodate the increased ACTH secretion to maintain glucocorticoid homeostasis. We propose that the most significant physiological effect of dietary phytoestrogens is to increase adrenal androgen (DHEA and DHEA-S) secretion as a consequence of decreased cortisol production and increased ACTH secretion. Previous studies suggested that the timing of adrenarche (when adrenal androgen production resumes at 6–10 yr of age) is influenced by estrogens (36). Precocious adrenarche in girls was associated with increased estradiol levels. Those findings and our present data raise the possibility that a phytoestrogen-rich diet may cause adrenarche to occur at a younger age and possibly promote masculinization, provided that the adrenal androgens are not converted to estrogens.
Our study brings together two somewhat controversial areas of biomedical research: 1) the effects of dietary phytoestrogens on human health, and 2) the role of the adrenal androgens DHEA and DHEA-S in human physiology. Despite relatively scant scientific evidence, the idea that DHEA promotes well-being has gained increasing popularity in recent years, and as a consequence, DHEA is readily available as an over the counter food supplement. Based upon our present findings, we postulate that a diet that includes a moderate amount of phytoestrogen-containing foods such as soy would be sufficient to raise endogenous DHEA and DHEA-S production to levels that may obviate the need for DHEA supplementation. Thus, it is possible that some of the actions of dietary phytoestrogens may be mediated via increased adrenal androgen production. It is of interest that DHEA is a precursor of androgenic and estrogenic endogenous sex steroids. Thus, by increasing DHEA production, dietary phytoestrogens may indirectly increase total estrogen and/or androgen levels. Therefore, it is possible that some of the estrogenic actions of dietary phytoestrogens may be mediated via their stimulation of adrenal androgen synthesis.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Present address: Mothers and Babies Research Centre, John Hunter
Hospital, Newcastle, New South Wales 2310, Australia.
3 Present address: California North Bay Fertility Associates, 1111
Sonoma Avenue, Santa Rosa, California 95405.
Received November 11, 1998.
Revised March 11, 1999.
Accepted March 31, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-hydroxylase/17,20 lyase): cloning of
human adrenal and testis cDNAs indicates the same gene is expressed in
both tissues. Proc Natl Acad Sci USA. 84:407–411.
5-4-isomerase in human
adrenals and gonads. Mol Endocrinol. 5:1147–1157.This article has been cited by other articles:
 |
|
 |
|
  V. Ajdzanovic, B. Sosic-Jurjevic, B. Filipovic, S. Trifunovic, M. Manojlovic-Stojanoski, M. Sekulic, and V. Milosevic Genistein-Induced Histomorphometric and Hormone Secreting Changes in the Adrenal Cortex in Middle-Aged Rats Experimental Biology and Medicine, February 1, 2009; 234(2): 148 - 156. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Baquedano, N. Saraco, E. Berensztein, C. Pepe, M. Bianchini, E. Levy, J. Goni, M. A. Rivarola, and A. Belgorosky Identification and Developmental Changes of Aromatase and Estrogen Receptor Expression in Prepubertal and Pubertal Human Adrenal Tissues J. Clin. Endocrinol. Metab., June 1, 2007; 92(6): 2215 - 2222. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-C. Chen, M. L Nagpal, D. M Stocco, and T. Lin Effects of genistein, resveratrol, and quercetin on steroidogenesis and proliferation of MA-10 mouse Leydig tumor cells J. Endocrinol., March 1, 2007; 192(3): 527 - 537. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. L. Dillingham, B. L. McVeigh, J. W. Lampe, and A. M. Duncan Soy Protein Isolates of Varying Isoflavone Content Exert Minor Effects on Serum Reproductive Hormones in Healthy Young Men J. Nutr., March 1, 2005; 135(3): 584 - 591. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J Liu, X-D Li, A Ora, P Heikkila, A Vaheri, and R Voutilainen cAMP-dependent protein kinase activation inhibits proliferation and enhances apoptotic effect of tumor necrosis factor-{alpha} in NCI-H295R adrenocortical cells J. Mol. Endocrinol., October 1, 2004; 33(2): 511 - 522. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Cifuentes, J. P. Advis, and S. A. Shapses Estrogen Prevents the Reduction in Fractional Calcium Absorption Due to Energy Restriction in Mature Rats J. Nutr., August 1, 2004; 134(8): 1929 - 1934. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. E. Wood, J. M. Cline, M. S. Anthony, T. C. Register, and J. R. Kaplan Adrenocortical Effects of Oral Estrogens and Soy Isoflavones in Female Monkeys J. Clin. Endocrinol. Metab., May 1, 2004; 89(5): 2319 - 2325. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Kallay, H. Adlercreutz, H. Farhan, D. Lechner, E. Bajna, W. Gerdenitsch, M. Campbell, and H. S. Cross Phytoestrogens Regulate Vitamin D Metabolism in the Mouse Colon: Relevance for Colon Tumor Prevention and Therapy J. Nutr., November 1, 2002; 132(11): 3490S - 3493. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. A. Whitehead, J. E. Cross, C. Burden, and M. Lacey Acute and chronic effects of genistein, tyrphostin and lavendustin A on steroid synthesis in luteinized human granulosa cells Hum. Reprod., March 1, 2002; 17(3): 589 - 594. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Nejaty, M. Lacey, and S. A. Whitehead Differing Effects of Endocrine-Disrupting Chemicals on Basal and FSH-Stimulated Progesterone Production in Rat Granulosa-Luteal Cells Experimental Biology and Medicine, June 1, 2001; 226(6): 570 - 576. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
